Monoclonal antibodies to human immunodeficiency virus and uses thereof

ABSTRACT

The present invention relates to novel monoclonal antibodies which may be used in the detection of Human Immunodeficiency Virus (HIV). These antibodies exhibit an unusually high degree of sensitivity, a remarkably broad range of specificity, and bind to novel shared, non-cross-reactive epitopes. In particular, the monoclonal antibodies of the present invention may be utilized to detect HIV-1 antigen and HIV-2 core antigen in a patient sample.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to novel monoclonal antibodies which maybe used in the detection of Human Immunodeficiency Virus (HIV). Theseantibodies exhibit an unusually high degree of sensitivity, a remarkablybroad range of specificity, and bind to novel shared, non-cross-reactiveepitopes. In particular, the monoclonal antibodies of the presentinvention may be utilized to detect HIV-1 antigen and HIV-2 core antigenin a patient sample.

2. Background Information

Acquired Immunodeficiency Syndrome (AIDS) is an infectious and incurabledisease transmitted through sexual contact from HIV infected individualsor by exposure to HIV contaminated blood or blood products. HIV-1includes the formerly named viruses Human T-cell Lymphotrophic VirusType III (HTLV III), Lymphadenopathy Associated Virus (LAV), and AIDSAssociated Retrovirus (ARV). HIV is a retrovirus related to a group ofcytopathic retroviruses, namely lentiviruses, on the basis ofmorphologic features, genomic organization, and nucleotide sequence(Gonda et al., Science (1985) 277:177-179; Stephan et al., Science(1986) 231:589-594; Korber, B. (ed.) et al., Human Retroviruses andAIDS. A Compilation and Analysis of Nucleic Acid and Amino AcidSequences. Published by Theoretical Biology and Biophysics, Los AlamosNational Laboratory, Los Alamos, N. Mex.; Reviewed in, Schochetman, G.and George, J. R., (1994) AIDS Testing. Springer-Verlag, N.Y., Berlin,Heidelberg). HIV is an enveloped virus containing several structuralproteins. Of particular relevance, the core of the virus is formed bycondensation of cleavage products from a highly processed gag-polpolyprotein precursor (Pr180gag-pol) which is cleaved into a polprecursor and a gag precursor (Pr55gag). Subsequently, the coreprecursor Pr55gag is cleaved into p17 (myristilated gag protein), p24(major structural protein), p7 (nucleic acid binding protein), and p9(proline-rich protein). The envelope contains two structural proteins,gp120 (envelope glycoprotein) and gp41 (transmembrane protein) which arecleavage products of the envelope polyprotein precursor, gp160.

The most common markers of HIV infection are antibodies against viralstructural proteins (Dawson, et.al., J. Infect. Dis. (1988) 157:149-155;Montagnier, et al. Virology (1985) 144:283-289; Barin, et al., Science(1985) 228:1094-1096; Schulz, T. F., et al., Lancet (1986) 2:111-112;Sarngadharan, et al., Science (1984) 224:506-508; Allan, et al., Science(1985) 228:1091-1093) and viremia in the form of detectable viral coreantigen (antigenemia) (Kessler, et. al., JAMA (1987) 258:1196-1199;Phair, JAMA (1987) 258:p1218; Allain, et al., The Lancet (1986)ii:1233-1236; Kenny, et al., The Lancet (1987) 1 (8532):565-566; Wall,et al., The Lancet (1987) 1(8532):p566; Stute, The Lancet (1987)1(8532):p566; Goudsmit, et al., The Lancet (1986) ii: 177-180; vonSydow,et al., Brit. Med. J. (1988) 296:238-240; Bowen, et al. Ann. of Int.Med. (1988) 108:46-48) or detectable viral nucleic acid (Mellors, etal., Science (1996) 272: 1167-1170; Saag, et al. Nat. Med. (1996) 2:625-629; Mulder, et al. J. Clin. Microbiol. (1994) 32:292-300; Zhang, etal., AIDS (1991) 5(6):675-681; Simmonds, et al., J. Virology (1990)64(2):864-872). For example, in the United States, screening of bloodand blood products by tests to detect antibody or antigen is mandated(Federal Food, Drug, and Cosmetic Act, 21 U.S.C.

301 et. seq., Public Health Service Act 42 U.S.C.

201 et. seq.). Nucleic acid testing recently has been implemented inorder to attain maximal reduction of the HIV seroconversion window(www.fda.gov). As a further example, various countries in Europe havebegun to evaluate and use tests that detect antibody and antigensimultaneously (Ly, et al. J. Clin. Microbiol. (2000) 38(6): 2459-2461;Gurtler, et al., J. Virol. Methods (1998) 75: 27-38; Weber, et al., J.Clin. Microbiol (1998) 36(8): 2235-2239; Courouce, et al., La Gazette dela Transfusion (1999) N° 155-Mars-Avril; Van Binsbergen, et al., J.Virol. Methods (1999) 82: 77-84), in addition to European implementationof nucleic acid testing. Serologic assays that combine antibody andantigen detection exhibit superior seroconversion sensitivity comparedto assays that detect only antibody, because detection of antigen, whichappears prior to antibody, reduces the seroconversion window. An earlyversion of an HIV combo assay is described in Gallarda, et al., 1992,WO93/21346, Assay for Detection of HIV Antigen and Antibody.

Within several weeks after infection with HIV, individuals generallyenter a clinical phase characterized by extensive viremia and acutesymptoms. During this period, prior to seroconversion, HIV p24 coreantigen can be detected transiently in serum or plasma specimens(antigenemia) (Devare, et al., (1990) In, Human Immunodeficiency Virus:Innovative Techniques. Monograph in Virology, J. L. Melnick (ed.),Basel, Karger, vol 18: 105-121; Kessler, et al. JAMA (1987 258:1196-1199; Phair, J. P., JAMA (1987) 258: p1218; Allain, et al. TheLancet (1986) ii: 1233-1236; Kenny, et al., The Lancet (1987) 1(8532):565-566; Wall, et al., The Lancet (1987) 1(8532): 566; Stute, R., TheLancet (1987) 1(8532): 566; Goudsmit, et al., The Lancet (1986) ii:177-180; vonSydow, et al., Brit. Med. J. (1988) 296: 238-240; Bowen, etal., Ann. of Int. Med. (1988) 108: 46-48). After seroconversion, thecore protein apparently is bound up by antibodies in circulating immunecomplexes, making core protein detection difficult and requiring immunecomplex disruption techniques (Schupbach, et al., AIDS (1996)10:1085-1090; Kageyama, et al., J. Virol. Methods (1988) 22: 125-131;Mathiesen, et al., J. Virol. Methods (1988) 22: 143-148; Steindl, etal., J. Immunol. Methods (1998) 217: 143-151; Euler, et al., Clin. Exp.Immunol. (1985) 59: 267-275; Gupta, et al., New Eng. J. Med. (1984) 310:1530-1531; Griffith, et al., J. Clin. Microbiol. (1995) 33: 1348-1350).After the initial viremic phase and throughout the remainder of thedisease, the virus generally establishes a steady state level (reviewedin Coffin, J. M. Science (1995) 267: 483-489).

Core proteins from isolates of HIV-1 group 0, HIV-1 group M, and HIV-2are antigenically similar because they share regions of amino acidsequence homology. Human (or mouse) immune polyclonal sera (i.e.,immunoglobulin) elicited against the core protein of one group or typewill cross react against the core protein of a different group or type(Clavel, et al., Science (1986) 233; 343-346; Guyader, et al., Nature(1987) 326: 662-669; Barin, et al., Lancet (1985) 2: 1387-1389; Kanki,et al., Science (1986) 232: 238-243; Kanki, et al., Science (1987) 236:827-831; Clavel, et al., Nature (1986) 324: 691-695; Hunt, et al., AIDSRes. Human Retroviruses (1997) 13: 995-1005; Gurtler, et al., J. Virol.Methods (1995) 51: 177-184; Mauclere, P. AIDS (1997) 11: 445-453).However, in contrast to human (or mouse) immune polyclonal sera, mouseor human monoclonal antibodies raised or elicited against the coreprotein of one HIV group or type may (Mehta, et al., U.S. Pat. No.5,173,399; Butman, et al., U.S. Pat. No. 5,210,181; Butman, et al., U.S.Pat. No. 5,514,541) or may not (Mehta, et al., U.S. Pat. No. 5,173,399;Butman, et al., U.S. Pat. No. 5,210,181; Butman, et al., U.S. Pat. No.5,514,541) react against the core protein of a different HIV group ortype. Often, however, neither cross-reactivity nor shared reactivity(Tijssen, 1993 In, Laboratory Techniques in Biochemistry and MolecularBiology. R. H. Burdon and P. H. van Knippenberg, eds. Vol. 15. Elsevier,Amsterdam) of mouse monoclonal antibodies have been considered or taught(Kortright, et al., U.S. Pat. No. 4,888,290; Kortright, et al., U.S.Pat. No. 4,886,742). In cases where HIV-1 and HIV-2 core proteins weredetected simultaneously (Butman, et al., U.S. Pat. No. 5,210,181;Butman, et al., U.S. Pat. No. 5,514,541), a combination of at least 3monoclonals were required, and the resulting quantitative sensitivityagainst HIV-1 core protein was much greater (50-fold) than for HIV-2core protein, indicating that the monoclonals identified cross-reactiveepitopes and not shared epitopes. Typically, monoclonal antibodiesdisplay a lower affinity against cross-reactive antigens (epitopes)(Karush, F. (1978) In, Comprehensive Immunology, ed. R. A. Good, S. B.Day, 5: 85-116. New York/London: Plenum; Mariuzza, et al., Rev. Biophys.Biophys. Chem. (1987) 16: 139-159; Tijssen, (1993) In, LaboratoryTechniques in Biochemistry and Molecular Biology. R. H. Burdon and P. H.van Knippenberg, eds. Vol. 15. Elsevier, Amsterdam) compared to theaffinity against the immunizing antigen (epitope) or shared epitope,resulting in less sensitivity toward the cross-reactive antigen.

Shared epitopes are not readily identified, particularly within proteinsof related but different sequence. A single amino acid change within anepitope can destroy or modify binding of a monoclonal antibody to thatepitope (Mariuzza, et al., Rev. Biophys. Biophys. Chem. (1987) 16:139-159). In addition, within proteins, amino acid changes (ordifferences) in sites outside of the epitope can change the epitope dueto changes in protein folding (Mariuzza, et al., Rev. Biophys. Biophys.Chem. (1987) 16: 139-159; Layer, et al., Cell (1990) 61: 553-556), thusaltering the binding of an antibody to the epitope. In this regard, thecore proteins of HIV-1 Group M, HIV-1 Group O, and HIV-2 are related butnot identical (Korber, ibid), and although it is known thatcross-reactive epitopes exist between HIV core proteins, it is neithercertain nor taught that shared epitopes are present.

The extensive genetic (and therefore antigenic) variability of HIV hasnot been predicted, although many scientific papers have sought tosupply explanations for the mechanism(s) of variability (Meyerhans, etal., Cell (1989) 58: 901-910; Wain-Hobson, Curr. Top. Microbiol.Immunol. (1992) 176:181-193; Holland, et al., Curr. Top. Micorbiol.Immunol. (1992) 176: 1-20; Gao, F. et al., Nature (1999) 397: 436-441;Sharp, et al., Biol. Bull. (1999) 196: 338-342; Robertson, et al.,Nature (1995) 374: 124-126; Zhu, J. Virol. (1995) 69: 1324-1327).Determination of HIV genetic (and therefore antigenic) variability hasrelied solely on many empirical observations that subsequently have ledto phylogenetic classification based on variation of HIV nucleic andamino acid sequence (Korber, ibid). Similarly, prediction of sharedepitopes between HIV (core) proteins cannot be made because (a) coreprotein sequences must first be discovered, (b) once discovered, geneticvariation provides added complexity and uncertainty to theidentification of shared epitopes and (c) epitope discovery andcharacterization are required to differentiate cross-reactive fromshared epitopes. Shared epitopes between HIV-1 Group M, HIV-1 Group O,and HIV-2 could not be determined until the discovery of HIV-1 Group Oin 1994 (Gurtler, et al., J. Virol. (1994) 68: 1581-1585; Haesevelde, etal., J. Virol. (1994) 68: 1586-1596; Charneau, et al., Virology (1994)205: 247-253).

The role of monoclonal antibody affinity for equivalent quantitativedetection of variable HIV core proteins generally has not been taught(Mehta, et al., U.S. Pat. No. 5,173,399; Gallarda, et al. WO93/21346;Zolla-Pazner, et al., U.S. Pat. No. 5,731,189; Mestan, et al., EP0519866A1; Butman, et al., U.S. Pat. No. 5,210,181; Butman, et al., U.S.Pat. No. 5,514,541; Kortright, et al., U.S. Pat. No. 4,888,290;Kortright, et al., U.S. Pat. No. 4,886,742). An average affinity for amonoclonal antibody elicited against a protein antigen is 4.5×10⁷ mol⁻¹(Mariuzza, et al., Rev. Biophys. Biophys. Chem. (1987) 16: 139-159;Karush, F. (1978) In, Comprehensive Immunology, ed. R. A. Good, S. B.Day, 5: 85-116. New York/London: Plenum). Additionally, immunizationstrategies to increase the probability of obtaining monoclonals againstshared epitopes have not been taught.

Only by combining two unpredictable features of monoclonal antibodies,affinity and shared reactivity, can one reasonably expect to obtainmonoclonal antibodies which can be used to detect equivalent amounts ofrelated but non identical HIV core proteins. Simple cross-reactivity ofmonoclonal antibodies is likely to be insufficient to achieve equivalentquantitative detection of HIV core proteins. Rather, shared reactivityin combination with high affinity is required to achieve the desiredresult. The affinity of a monoclonal for a related core protein may besubstantially lower than that determined with the immunizing coreprotein. In that case, the epitope is most likely cross-reactive and theaffinity of the antibody for the cross-reactive epitope may severelylimit the utility of the antibody for detection of diagnosticallyrelevant (i.e., 25 pg p24/ml serum or plasma, Courouc•, et al., LaGazette de la Transfusion (1999) N° 155-Mars-Avril) concentrations ofthe cross reactive core protein.

There are currently no known descriptions of immunoassays using only 2monoclonal antibodies to achieve equivalent quantitative detection ofHIV-1 Group M, HIV-1 Group O, and HIV-2 core proteins. Thus, such animmunoassay is certainly desirable. Two or more monoclonals incombination with polyclonal sera (immunoglobulin) have provided thebasis for immunoassays to detect HIV-1 core protein or simultaneouslyHIV-1 and HIV-2 core proteins (Mehta, et al., U.S. Pat. No. 5,173,399;Butman, et al., U.S. Pat. No. 5,210,181; Butman, et al., U.S. Pat. No.5,514,541; Kortright, et al., U.S. Pat. No. 4,888,290; Kortright, etal., U.S. Pat. No. 4,886,742; Gallarda, et al. WO93/21346). Thus, inview of the above, previous literature fails to (a) describe or teachimmunoassay restricted to two monoclonals for equivalent quantitativedetection of HIV-1 Group M and HIV-2 core proteins, (b) describe orteach immunoassays restricted to two monoclonal antibodies forequivalent quantitative detections of HIV-1 group M, HIV-1 group O, andHIV-2 core proteins, (c) teach methods to overcome monoclonal affinitybarriers recognizing cross-reactive antigens leading to non-equivalentdetection of HIV-1 group M, O, and HIV core proteins, and (d) highaffinity monoclonal antibodies against shared-epitopes as the methodsand means to detect diagnostically relevant and equivalent amounts ofnon-identical core proteins from HIV-1 group M, HIV-2 group O, andHIV-2.

All U.S. patents, patent applications and publications referred toherein are hereby incorporated in their entirety by reference.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies and methods ofusing these antibodies in the detection of Human Immunodeficiency VirusType 1 (Groups M and O) and Type 2, the etiologic agents of AcquiredImmunodeficiency Syndrome (AIDS), in serum, plasma, or other bodilyfluids. In particular, the invention encompasses diagnostic methods thatemploy compatible, high affinity, unique mouse monoclonal antibodiesidentifying non-cross-reactive, shared epitopes in order to detectequivalent amounts of HIV-1 core protein (p24) and HIV-2 core protein(p26). Such antibodies also may be used in assays which detect HIVantigen and in combination assays that simultaneously detect HIV antigenand HIV antibody. In a preferred embodiment of the present invention,only two complementary, high affinity, broadly specific mouse monoclonalantibodies are required to detect equivalent amounts of core proteinsfrom HIV-1 Group M, HIV-1 Group O, and HIV-2.

The monoclonal antibodies of the present invention have high affinities(Keq values) sufficient to detect diagnostically relevant femtomolarquantities of HIV core protein; however, they also possess broadspecificity (i.e., shared-reactivity) for detection of equivalentquantities of related, but nonidentical, core proteins from HIV-1 GroupM, HIV-1 Group O, and HIV-2.

In particular, the present invention encompasses monoclonal antibodieswhich specifically bind to Human Immunodeficiency Virus-1 groups O and Mprotein p24 and Human Immunodeficiency Virus-2 protein p26. Thesemonoclonal antibodies are, for example, 120A-270, 115B-151, 103-350,115B-303., 117-289, and 108-394. The present invention also includes thehybridomas that produce these antibodies.

Furthermore, the present invention also encompasses a method fordetecting the presence of one or more antigens selected from the groupconsisting of HIV-1 antigen and HIV-2 antigen, in a test samplesuspected of containing one or more of the antigens. The methodcomprises the steps of: a) contacting the test sample with at least onemonoclonal antibody (e.g., 120A-270) which specifically binds to sharedepitopes on Human Immunodeficiency Virus-1 protein p24 and HumanImmunodeficiency Virus-2 protein p26 for a time and under conditionssufficient for the formation of antibody/antigen complexes; and b)detecting the complexes, presence of the complexes indicating presenceof at least one antigen selected from the group consisting of HIV-1antigen and HIV-2 antigen, in the test sample. The monoclonal of step(a) may be, for example, any one of the monoclonal antibodies describedherein. It may or may not be labeled. Preferably, only one monoclonalantibody is contacted with the test sample.

The present invention also includes a method for simultaneouslydetecting the presence of one or more antigens selected from the groupconsisting of HIV-1 antigen and HIV-2 antigen, in a test samplesuspected of containing one or more of the antigens. The methodcomprises the steps of: a) contacting the test sample with at least onemonoclonal antibody which specifically binds to Human ImmunodeficiencyVirus-1 protein 24 and Human Immunodeficiency Virus-2 protein p26 for atime and under conditions sufficient for the formation ofantibody/antigen complexes; b) adding a conjugate to the resultingantibody/antigen complexes for a time and under conditions sufficient toallow the conjugate to bind to the bound antigen, wherein the conjugatecomprises an antibody attached to a signal generating compound capableof generating a detectable signal; and c) detecting the presence ofantigen which may be present in the test sample by detecting a signalgenerated by the signal-generating compound, presence of the signalindicating presence of at least one antigen selected from the groupconsisting of HIV-1 antigen and HIV-2 antigen in the test sample. The atleast one monoclonal antibody of step (a) may be, for example, 120A-270,115B-151, 117-289, 103-350, 108-394 or 115B-303. Preferably, onemonoclonal antibody is used, in particular, 120A-270. The antibody ofstep (b) of the conjugate may be, for example, 120A-270, 115B-151,117-289, 103-350, 108-394 or 115B-303, and is preferably 115B-151.Preferably, monoclonal antibody 120A-270 (or 117-289) and monoclonalantibody 115B-151 are used as a pair, whether 120A-270 (or 117-289) ison the solid phase or is present in the conjugate, or whether 115B1-151is on the solid phase or is present in the conjugate.

Moreover, the present invention also encompasses a method for detectingthe presence of one or more antigens selected from the group consistingof HIV-1 antigen and HIV-2 antigen, in a test sample suspected ofcontaining one or more of these antigens, comprising the steps of: (a)simultaneously contacting: 1) at least one monoclonal antibody, whichspecifically binds to HIV-1 p24 antigen and HIV-2 p26 antigen, bound toa solid support, 2) the test sample, and 3) an indicator reagentcomprising an antibody which specifically binds to HIV-1 antigen andHIV-2 antigen to which a signal generating compound is attached, to forma mixture; (b) incubating the mixture for a time and under conditionssufficient to form antibody/antigen/antibody complexes; (c) detectingthe presence of a measurable signal generated by the signal-generatingcompound, presence of the signal indicating presence of one or moreantigens in said test sample selected from the group consisting of HIV-1antigen and HIV-2 antigen. The at least one monoclonal antibody of step(a) may be, for example, 120A-270, 115B-151, 117-289, 108-394, 115B-303or 103-350, and is preferably 120A-270. The antibody of the conjugate ofstep (b) may be, for example, 120A-270, 115B-151, 117-289, 108-394,115B-303 or 103-350, and is preferably 115B-151. Again, it is importantto note that any one or more monoclonal antibodies of the presentinvention may be used on the solid phase in connection with any othermonoclonal antibody of the invention (in the conjugate or solutionphase). Certain pairs of monoclonal antibodies are preferred, however,and it is preferable to have only one monoclonal antibody on the solidphase.

The present invention also includes a kit for determining the presenceof one or more antigens selected from the group consisting of HIV-1antigen and HIV-2 antigen in a test sample comprising: (a) at least onemonoclonal antibody which which specifically binds to HumanImmunodeficiency Virus-1 protein p24 and Human Immunodeficiency Virus-2protein p26; and (b) a conjugate comprising an antibody attached to asignal-generating compound capable of generating a detectable signal.The at least one monoclonal antibody of (a) may be, for example,120A-270, 115B-151, 117-289, 108-394, 115B-303, or 103-350, and ispreferably 120A-270. The antibody of (b) may be, for example, 120A-270,115B-151, 117-289, 108-394, 115B-303, or 103-350, and is preferably115B-151.

The present invention also includes a diagnostic reagent comprising atleast one monoclonal antibody selected from the group consisting of120A-270, 115B-151, 117-289, 103-350, 108-394 and 115B-303.

Additionally, the present invention encompasses isolated epitopes orpeptides having the amino acid sequences shown in SEQ ID Nos: 1-6.

The present invention also includes methods of simultaneously detectingboth antigen and antibody to HIV-1 and/or HIV-2 in a patient sample. Onesuch method involves detecting 1) one or more antibodies selected fromthe group consisting of HIV-1 antibody and HIV-2 antibody, and 2) one ormore antigens selected from the group consisting of HIV-1 antigen andHIV-2 antigen, in a test sample suspected of containing one or more ofthe antibodies and one or more of said antigens, comprising the stepsof: a) contacting the test sample with at least one HIV-1 antigen whichbinds to HIV-1 antibody for a time and under conditions sufficient forthe formation of HIV-1 antigen/HIV-1 antibody complexes; b) detectingthe HIV-1 antigen/HIV-1 antibody complexes, presence of the complexesindicating presence of HIV-1 antibody in the test sample; c) contactingthe test sample with at least one HIV-2 antigen which binds to HIV-2antibody for a time and under conditions sufficient for the formation ofHIV-2 antigen/HIV-2 antibody complexes; d) detecting the HIV-2antigen/HIV-2 antibody complexes, presence of the complexes indicatingpresence of HIV-2 antibody in the test sample; e) contacting the testsample with at least one monoclonal antibody which specifically binds toHuman Immunodeficiency Virus-1 protein p24 and Human ImmunodeficiencyVirus-2 protein p26 for a time and under conditions sufficient for theformation of antibody/antigen complexes; and f) detecting the complexes,presence of the complexes indicating presence of at least one antigenselected from the group consisting of HIV-1 antigen and HIV-2 antigen,in the test sample. Again, it is preferable to utilize certain pairs ofmonoclonal antibodies in connection with HIV-1 and HIV-2 antigendetection (e.g., 120A-270 and 115B-151).

Another method enocompassed by the present invention involvesdetecting 1) one or more antibodies selected from the group consistingof HIV-1 antibody and HIV-2 antibody, and 2) one or more antigensselected from the group consisting of HIV-1 antigen and HIV-2 antigen,in a test sample suspected of containing one or more of the antibodiesand one or more of the antigens, comprising the steps of: a) contactingthe test sample with at least one HIV-1 antigen which specifically bindsto HIV-1 antibody for a time and under conditions sufficient for theformation of HIV-1 antigen/HIV-1 antibody complexes; b) adding aconjugate to the resulting HIV-1 antigen/HIV-1 antibody complexes for atime and under conditions sufficient to allow the conjugate to bind tothe bound antibody, wherein the conjugate comprises an antigen attachedto a signal-generating compound capable of generating a detectablesignal; c) detecting HIV-1 antibody which may be present in the testsample by detecting a signal generated by the signal-generatingcompound, presence of the signal indicating presence of HIV-1 antibodyin the test sample; d) contacting the test sample with at least oneHIV-2 antigen which specifically binds to HIV-2 antibody for a time andunder conditions sufficient for the formation of HIV-2 antigen/HIV-2antibody complexes; e) adding a conjugate to the resulting HIV-2antigen/HIV-2 antibody complexes for a time and under conditionssufficient to allow the conjugate to bind to the bound antibody, whereinthe conjugate comprises an antigen attached to a signal generatingcompound capable of generating a detectable signal; f) detecting HIV-2antibody which may be present in the test sample by detecting a signalgenerated by the signal-generating compound, presence of the signalindicating presence of HIV-2 antibody in the test sample; g) contactingthe test sample with at least one monoclonal antibody which specificallybinds to Human Immunodeficiency Virus-1 protein 24 and HumanImmunodeficiency Virus-2 protein p26 for a time and under conditionssufficient for the formation of antibody/antigen complexes; h) adding aconjugate to the resulting antibody/antigen complexes for a time andunder conditions sufficient to allow the conjugate to bind to the boundantigen, wherein the conjugate comprises an antibody attached to asignal-generating compound capable of generating a detectable signal;and i) detecting presence of antigen which may be present in said sampleby detecting a signal generated by the signal-generating compound,presence of the signal indicating presence of at least one antigenselected from the group consisting of HIV-1 antigen and HIV-2 antigen inthe test sample. Again, the preferred pairs of monoclonal antibodieswhich may be used in the assay are described above; however, other pairsmay also be utilized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a illustrates amino acid sequence alignment of p24 from HIV-1group M and HIV-1 group O. Structural features (e.g., helices A-J)determined from group M p24 structure are shown above the sequencealignment. Synthetic group M and group 0 p24 peptides for mappingstudies were designed and labeled according to group M or O sequence andnumbering respectively.

FIG. 1 b illustrates amino acid sequence alignment of p24 from HIV-1group M, HIV-1 group O, and HIV-2 p26.

FIGS. 2 a and 2 b illustrate the binding of monoclonal antibodies103-350, 117-289, 115-303, 120A-270, and 115B-151 to p24 syntheticpeptides.

FIG. 3 illustrates the location of deletion clones derived from p24 ofHIV-1 group M and O.

FIG. 4 illustrates the results of Western blots used to map binding ofmonoclonal antibodies 115B-151 and 108-394 to regions of p24.

FIG. 5 summarizes HIV-1 p24 epitopes recognized by p24 monoclonalantibodies.

FIG. 6 illustrates HIV-1 group M p24 quantitative sensitivity achivedusing 120A-270 on a solid phase and 115B-151 in solution phase.

FIG. 7 illustrates HIV-1 group O p24 quantitative sensitivity achievedusing 120A-270 on a solid phase and 115B-151 in solution phase.

FIG. 8 illustrates HIV-2 p26 quantitative sensitivity achieved using120A-270 on a solid phase and 115B-151 in solution phase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel monoclonal antibodies to HIV-1protein p24 and HIV-2 protein p26, methods for using these monoclonalantibodies, and kits containing these antibodies. More specifically, thepresent invention relates to monoclonal antibodies referred to herein as120A-270 (e.g., clone 108), 115B-151 (e.g., clone 423), and 117-289(e.g., clone 555).

Additionally, the present invention includes monoclonal antibodiesreferred to herein as 103-350 (e.g., clone 474), 108-394 (e.g., clone470) and 115B-303 (e.g., clone 620).

The present invention not only includes the monoclonal antibodiesreferred to above but also includes the novel hybridoma cell lines whichproduce these antibodies. More specifically, the cell line ATCC HB______ produces monoclonal antibody 120A-270, the cell line ATCC HBproduces monoclonal antibody 115B-151, the cell line ATCC HB ______produces monoclonal antibody 117-289, the cell line ATCC HB ______produces monoclonal antibody 103-350, the cell line ATCC HB ______produces monoclonal antibody 108-394, and the cell line ATCC HB ______produces monoclonal antibody 115B-303. The cell lines producing theantibodies were deposited with the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110 under the terms of theBudapest Treaty on ______, 2000 and were accorded the ATCC numbers notedabove.

The monoclonal antibodies of the present invention or fragments thereofmay be used in immunoassays for the detection of HIV-1 (Groups M and O)and HIV-2, simultaneously. (For purposes of the present invention, a“fragment” is defined as a subunit of the monoclonal antibody whichreacts in the same manner, functionally, as the full antibody withrespect to binding properties.) In particular, when monoclonalantibodies 120A-270 and 115B-151, or monoclonal antibodies 117-289 and115B-151 are used in combination in an immunoassay, for example, in asandwich assay, one may minimally detect core antigen (p24) fromsubtypes A, B, C, D, E, F, G and O of HIV-1 groups M and O, and HIV-2core antigen (p26) in a patient sample. In fact, less than. 25 picogram(i.e., picogram core antigen/ml of serum or plasma) quantities of theHIV-1 p24 antigen and HIV-2 p26 antigen may be detected using thecombinations of monoclonal antibodies described above. Thus, themonoclonal antibodies of the present invention have a high degree ofsensitivity as well as broad specificity. In particular, the uniqueproperty of these antibodies is that they recognize related, butnon-identical, core antigens with approximately equivalent affinity(i.e., equivalent quantitative sensitivity), indicating that theyrecognize unpredictable shared epitopes, and thus exhibit sharedreactivity, rather than typical and expected cross-reactive epitopes andthus exhibiting cross-reactivity. (For purposes of the presentinvention, “cross-reactivity” is defined as the binding of an antibodyto structurally different determinants on different antigens. Antibodyaffinity for a cross-reactibe epitope (i.e., antigen) is lower than thatfor the immunogenic epitope (i.e., antigen) or shared epitope. “Sharedreactivity” is defined as the binding of an antibody to structurallyidentical determinants on different antigens. Antibody affinity for ashared epitope is equivalent to the affinity for the immunogenic epitope(i.e., immunogen).) It should also be noted that the pairs of monoclonalantibodies are compatible, that is, each monoclonal antibody of the pairmaps to a different epitope or antigenic determinant on the coreprotein(s). Binding of one antibody of the pair does not interfere withbinding of the second antibody of the pair.

In one embodiment of the invention, the preferred embodiment, monoclonalantibody 120A-270 or a fragment thereof is coated onto a solid phase(e.g., a microparticle, a microtiter well, a bead, etc.); however,115B-151 or 117-289 may also be used or fragments thereof. The testsample is then contacted with the monoclonal antibody or fragmentthereof such that, if p24 antigen or p26 antigen is present in thepatient sample, antibody/antigen complexes are then formed as a firstmixture. (For example, both monoclonal antibody/p24 antigen andmonoclonal antibody/p26 antigen complexes may be formed if the patienthas both HIV-1 and HIV-2.) One then adds a conjugate comprising (a) aprobe antibody, for example, monoclonal antibody 115B-151 (which bindsan epitope distinct from and compatible with the epitope bound by120-270) attached to (b) a signal-generating compound.Antibody/antigen/antibody probe complexes are then formed as a secondmixture. HIV-1 and/or HIV-2 antigen is then detected in the sample bydetecting the presence of the signal generated and thus theantibody/antigen/antibody probe complexes. The amount of antigen(s) inthe test sample may also be calculated, as the signal generated isproportional to the amount of antigen in the sample.

Another manner of detecting the complexes formed is to utilize aconjugate comprising a third antibody attached to a signal-generatingcompound. In particular, once the antibody/antigen/antibody complexesdescribed above have formed (i.e., the latter antibody being the 2^(nd)antibody which is unlabelled), one may then add a conjugate which bindsto the “2^(nd)” unlabelled antibody in solution. The conjugate maycomprise, for example, an antigen or anti-antibody capable of binding tothe bound second antibody (e.g., anti-115B-151 antibody or an antibodyto the probe antibody) attached to a signal-generating compound capableof generating a detectable signal. Detection of the signal thusindicates presence of the complexes and thus presence of the antigen inthe sample. The signal generated is actually proportional to the amountof antigen present in the sample. (See, e.g., U.S. Pat. No. 6,015,662.)The design of the assay is dependent upon the affinities andspecificities of the antibodies used, accuracy of results obtained,convenience, the nature of the solid phase, etc. (See U.S. Pat. No.5,104,790 for a discussion of different antigen assay formats.)Additionally, it should also be noted that the initial capture antibodyused in the immunoassay may be covalently or non-covalently (e.g.,ionic, hydrophobic, etc.) attached to the solid phase. Linking agentsfor covalent attachment are known in the art and may be part of thesolid phase or derivatized to it prior to coating. Examples of solidphases used in immunoassays are porous and non-porous materials, latexparticles, magnetic particles, microparticles, beads, membranes,microtiter wells and plastic tubes. The choice of solid phase materialand method of labeling the antigen or antibody present in the conjugate,if desired, is determined based upon desired assay format performancecharacteristics.

As noted above, the conjugate (or indicator reagent) will comprise anantibody (or perhaps anti-antibody, depending upon the assay), attachedto a signal-generating compound or label. This signal-generatingcompound or “label” is in itself detectable or may be reacted with oneor more additional compounds to generate a detectable product. Examplesof signal-generating compounds include chromogens, radioisotopes (e.g.,125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g.,acridinium), particles (visible or fluorescent), nucleic acids,complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluro-, or iumo-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful.

Another type of assay in which the present monoclonal antibodies may beutilized involves simultaneously contacting: 1) one monoclonal antibody(bound to a solid support), 2) the test sample and 3) an indicatorreagent comprising a monoclonal antibody or fragment thereof (e.g.,115B-151, which specifically binds to HIV-1 and HIV-2 antigen) to whicha signal generating compound is attached, to form a mixture. The mixtureis then incubated for a time and under conditions sufficient to formantibody/antigen/antibody complexes. The presence, if any, of HIV-1and/or HIV-2 antigen present in the test sample and captured on thesolid phase is determined by detecting the measurable signal generatedby the signal-generating compound. The amount of antigen present in thetest sample is proportional to the signal generated. In this assay orthose described above, the monoclonal antibodies of the presentinvention may be used either as the capture phase or as part of theindicator reagent in solution (i.e., the reagent comprising an antibodyand a signal-generating compound). Such diagnostic procedures, includingthose described above and below, are well-known in the art (seeImmunological Methods, Vols. I and II, 1979 and 1981, Eds., Lefkovitsand Pernis, Academic Press, New York; Monoclonal Antibodies, 1982, eds.,Kennett et al., Plenum Press, New York; and Handbook of ExperimentalImmunology, 1978, ed., Weir, Blackwell Scientific Publications, St.Louis, Mo.).

It should be noted that the monoclonal antibodies of the presentinvention preferably may be used either alone, as a single captureantibody, or alone as a single probe and/or conjugated antibody.However, they may also be used in pairs or in trios in the assaysdescribed above. Further, combinations of the monoclonal antibodies ofthe present invention (and fragments thereof) may be used with othermonoclonal antibodies that have specificities for epitopes of HIV-1and/or HIV-2, other than the epitope specificities of the monoclonalantibodies of the present invention. Thus, the present monoclonalantibodies may act as components in a mixture or “cocktail” of HIV-1and/or HIV-2 antibodies. Thus, for example, this cocktail can include amonoclonal antibody of the present invention which detects p24 of HIV-1and p26 of HIV-2 (e.g., 120A-270) and a monoclonal antibody whichdetects a HIV envelope antigenic determinant in the transmembraneprotein or extracellular glycoprotein. In this manner, one may be ableto detect several antigenic determinants from different proteins of oneor more viruses (e.g., HIV-1 and HIV-2) simultaneously.

Also, it should be noted that the monoclonal antibodies of the presentinvention may be utilized in a combination assay which detects: 1)antigens, such as those described above (e.g., p24 and p26) and 2)antibodies to HIV (by use of, for example, envelope antigens (e.g.,HIV-1 group M and 0 gp41 and HIV-2 gp36). Any such combination assay,which utilizes the monoclonal antibodies of the present invention, isconsidered to be within the scope of the invention.

Examples of biological fluids which may be tested by the aboveimmunoassays include plama, serum, cerebrospinal fluid, saliva, tears,nasal washes or aqueous extracts of tissues and cells. The test samplesmay also comprise inactivated whole virus or partially purified orrecombinant p24 or p26 antigen.

It should also be noted that the above-referenced monoclonal antibodiesmay be used, when appropriately labeled, as competitive probes againstHIV-1 and -2 core antibodies in serum samples for binding torecombinantly-derived HIV-1 p24 and HIV-2 p26.

Additionally, the monoclonal antibodies of the present invention orfragments thereof may be used in detection systems using fixed cells orfixed tissues, with appropriate labeling of each monoclonal antibody. Inparticular, the tissue sample is contacted with a conjugate comprising asignal-generating compound attached to one of the monoclonal antibodiesof the present invention in order to form a mixture. The mixture is thenincubated for a time and under conditions sufficient forantigen/antibody complexes to form. The presence of antigen present inthe sample is determined by detecting the signal generated. Theantibodies may also be utilized for purifying HIV-1 p24 antigen andHIV-2 p26 antigen by, for example, affinity chromatography.

Furthermore, the antibodies of the invention may be bound to matricesand used for the affinity purification of specific HIV-1 and/or HIV-2antigens from, for example, cell cultures, or biological tissues such asblood and liver. The monoclonal antibodies, for example, may be attachedto or immobilized on a substrate or support. The solution containing theHIV antigenic determinants is then contacted with the immobilizedantibody for a time and under conditions suitable for the formation ofimmune complexes between the antibody and polypeptides containing thep24 and p26 determinants. Unbound material is separated from the boundimmune complexes. The complexes or antigenic fragments are thenseparated from the support.

One or more of the monoclonal antibodies of the present invention, andpreferably the pairs suggested above, is particularly suitable for usein the form of a kit. The kit may comprise one or more containers suchas vials or bottles, with each container containing a pair of themonoclonal antibodies, or as cocktails of monoclonal antibodies. Thesekits may also contain vials or containers of other reagents needed forperforming the assay, such as washing, processing and indicatorreagents.

Additionally, the present invention also includes a vaccine comprisingone or more of the monoclonal antibodies of the present invention and apharmaceutically acceptable adjuvant (e.g., Freund's adjuvant) which canbe administered to HIV-infected individuals (i.e., passiveimmunization). Furthermore, the monoclonal antibodies of the presentinvention can serve prophylactically for administration to non-infected,high-risk individuals, such as health care workers.

It should also be noted that the monoclonal antibodies of the presentinvention may also serve as research tools for epitope mapping of HIVproteins p24 and p26. Further, it should be noted that not only do themonoclonal antibodies of the present invention bind to proteins andprotein precursors of HIV clinical isolates which contain the targetedregion or regions of antigenic determinants, in addition, the antibodiesbind to recombinant proteins and synthetic analogues of the proteinswhich contain the antigenic determinant(s). Thus, for example, themonoclonal antibodies of the present invention may be used in bindingexperiments involving recombinant proteins and synthetic analogues ofp24 of HIV-1 and p26 of HIV-2.

Additionally, antibodies of the present invention which are unlabeledmay be used in agglutination assays or can be used in combinantion withlabeled antibodies that are reactive with the monoclonal antibody, suchas antibodies specific for immunoglobulin.

The present invention also comprises a method for treating a mammalinfected with HIV-1 and/or HIV-2 comprising administering to a mammal,in need of such treatment, an effective amount of one of more of themonoclonal antibodies of the present invention in the form of apharmaceutical composition, as described directly below. Apharmaceutically effective amount means any amount of the compoundwhich, when incorporated in the pharmaceutical composition, will beeffective to inhibit HIV replication and thereby treat AcquiredImmunodeficiency Syndrome (AIDS) but less than an amount which would betoxic to the subject.

Additionally, the present invention encompasses pharmaceuticalcompositions comprising one or more of the monoclonal antibodies of thepresent invention and a pharmaceutically acceptable carrier. Apharmaceutical carrier is any compatible, non-toxic substance suitableto deliver one or more monoclonal antibodies to the patient. Forexample, sterile water, alchohol, fats, waxes and inert solids may beused as carriers. The composition may also contain monoclonal antibodieswhich bind to proteins or glycoproteins of HIV other than p24 and/orp26. Further, the pharmaceutical composition may be administered aloneor in conjunction with other anti-retroviral agents. (See Mitsuya etal., Nature 325:773-778 (1987).) The pharmaceutical compositions of thepresent invention may be administered either orally or parenterally(i.e., subcutaneously, intramuscularly or intravenously).

Further, it should be noted that one or more of the monoclonalantibodies of the present invention may be used to generate chimericantibodies for therapeutic use, for example, or as assay controls orcalibrators.

Since all of the monoclonal antibodies of the present invention bindboth to p24 of HIV-1 and to p26 of HIV-2, as evidenced by the datapresented in Table 5, for example, any one of more of the monocloanlantibodies may be used in the diagnostic assays, kits, compositions andmethods described above. Certainly those with the strongest bindingspecificities and capabilities (with respect to p24 and p26) arepreferred.

The present invention may be illustrated by the use of the followingnon-limiting examples:

EXAMPLE 1 Immunogen Selection

The immunization strategies included HIV-1 group O and HIV-1 group Mantigens to drive the immune response toward recognition of both sharedepitopes within the core antigens of both groups of HIV-1. Threedifferent HIV-1 immunogens were used in various combinations to developan anti-HIV-1 p24 response in the animal host. Two HIV-1 group Mantigens manufactured at Abbott Laboratories (Abbott Park, Ill.) werederived from denatured whole viral lysates while native HIV-1 group Mp24 (p24M) protein was purified from the viral lysates. The thirdimmunogen was a recombinant p24 antigen (rp24-O) derived from the gaggene of HIV-1 group O isolate HAM112. The p24 gene from HAM112 wascloned into the lambda PL vector and expressed in E. coli. Theconstruction, scale up and purification of the recombinant antigen wereperformed according to published methods for recombinant proteins madein E. coli. (Seetharam, R. and Sharma, S. K., (eds), 1991. ‘Purificationand Analysis of Recombinant Proteins’, Marcel Dekker. New York, N.Y.)Verification of the amino acid sequence against published resultsconfirmed integrity of the product. (Van den Haesevelde et al., 1994.Genomic cloning and complete sequence analysis of a highly divergentAfrican human immunodeficiency virus isolate. J. Virol. 68:1586.)

EXAMPLE 2 Immunization of Mice

The animal models selected for hybridoma development were three strainsof mice, the CAf1, the RBf/dn, and the BALB/c. The mice were females,age 6-8 weeks old, purchased from Jackson Laboratory (Bar Harbor, Me.).In order to produce anti-HIV-1 p24 monoclonal antibodies with highaffinity, two different immunization strategies were utilized.Hybridomas secreting anti-p24 monoclonal antibodies (Mab) 103-350-474,108-394-470, 115B-303-620, 115B-151-423, and 117-289-555 were producedfrom mice which were immunized twice with rp24-O or p24-M or mixture ofboth rp240 and p24M subcutaneously (s.c.) or intramuscularly (i.m.). Themice were rested for 4-12 months for affinity maturation and boostedintrasplenically (i.s.) with immunogen three days prior to fusion.120A-270-108 was produced from a BALB/c mouse that was hyperimmunizedweekly six times with low dosage of purified native p24-M givenalternatively between intraperitoneal (i.p.) administration andsubcutaneous administration.

The immunization procedures are described in detail as follows:

Hybridoma 103-350-474 was produced from cell fusion #103. On day 1, CAF1mouse #1555 received 10 ug of rp24O antigen in 0.2 ml of Freund'sComplete Adjuvant (CFA) (Difco Laboratories, Detroit, Mich.) givensubcutaneously. On day 56, the mouse received 10 ug of rp240 antigen in0.2 ml of Incomplete Freund's Adjuvant (IFA) (Difco Laboratories,Detroit, Mich.) given intramuscularly (i.m.). On day 74, the mouse wasbled for assessment of anti-HIV-1 antibody titer by indirectenzyme-linked immunoassay (EIA). On day 186, the mouse was boosted i.s.with 25 ug of HIV-1 group M viral lysate in normal saline.

Hybridoma 108-394-470 was produced from cell fusion #108. On day 1, CAF1mouse #1556 received 10 ug of rp240 antigen in 0.2 ml of CFA given s.c.On day 56, the mouse received 10 ug of rp240 antigen in 0.2 ml of IFAgiven i.m. On day 74, the mouse was bled for assessment of anti-HIV-1antibody titer by indirect EIA. On day 270, the mouse was boosted i.s.with 45 ug of purified native HIV-1 group M p24 in normal saline.

Hybridomas 115B-151-423 and 115B-303-620 were produced from cell fusion#115B. On day 1, CAF1 mouse #1563 received 10 ug of rp240 antigen in 0.2ml of CFA given s.c. On day 210, the mouse received mixture of 10 ug ofrp240 and 10 ug of p24M in 0.2 ml of IFA given s.c.

On day 235, the mouse was bled for assessment of anti-HIV-1 antibodytiter by indirect EIA. On day 375, the mouse was boosted i.s. with amixture of 10 ug of rp240 and 10 ug of p24M in normal saline.

Hybridoma 117-289-555 was produced from cell fusion #117. On day 1,RBf/dn mouse #1545 received 10 ug of rp240 antigen in 0.2 ml of CFAgiven s.c. On day 56, the mouse received mixture of 10 ug of rp240 in0.2 ml of IFA given i.m. On day 74, the mouse was bled for assessment ofanti-HIV-1 antibody titer by indirect EIA. On day 392, the mouse wasboosted i.s. with a mixture of 45 ug of rp240 and purified native p24Min normal saline. Hybridoma 120A-270-108 was produced from cell fusion#120A of a hyperimmunized BALB/c mouse #7 which received 0.2 ml ofimmunogen containing 10 ug of purified native p24M antigen and 4 ug ofS. typhimurium extract (RIBI Immunochemicals, RiBi Immuno Chem Research,Hamilton, Mont.) given i.p. on day 1, s.c. on day 7, and i.p. on day 14.On day 21, the mouse was bled for assessment of anti-HIV-1 p24 antibodytiter by indirect EIA. On days 28, 35, and 42, the mouse received 5 ugof purified native p24M antigen in 0.2 ml of RIBI adjuvant given s.c.,i.p., and s.c., respectively. On day 49, the mouse was bled a secondtime for assessment of anti-HIV-1 antibody titer by indirect EIA. On day77, three days before fusion, the mouse was boosted i.s. with 50 ug ofpurified native p24M antigen in normal saline.

EXAMPLE 3 Assessment of Anti-p24 Antibody Titer of the Immunized Mice

Indirect binding and direct sandwich enzyme-linked immunoassays (EIA)were used to assess anti-HIV-1 antibody titers from the immunized mice.(Direct sandwich EIA was performed with a limited amount of core antigento detect only high affinity antibodies.) Sera from naïve or immunizedmice were serially diluted in 10 mM sodium phosphate buffer (PBS), pH7.4, containing 5% bovine serum albumin (BSA) and 0.03% sodium azide aspreservative. The detailed assay procedures are described below. Theassessment of anti-p24 antibody titers from the immunized mice is shownin Table 1a from the indirect binding EIA and Table 1b from the directsandwich EIA. TABLE 1a Assessment of anti -p24 antibody titers byindirect EIA A490 nm reading Sera BSA Fusion # Animal ID dilution rp24-Op24-M control 103 CAf1 #1555 1/24,300 1.219 1.156 0.008 108 CAf1 #15561/24,300 1.157 1.109 0.005 117 RBf/dn #1545 1/900 0.462 0.600 0.019 120ABALB/c #7 1/100,000 NT 1.709 0.025 Pre-bled ms serum 1/900 0.036 0.0930.018

TABLE 1b Assessment of anti -p24 antibody titers by direct sandwich EIAA490 nm reading M O Fusion # Animal ID Sera dilution lysate lysate 115BCAf1 #1563 1/8,100 0.540 0.401 120A BALB/c #7 1/1,000 1.579 0.762Pre-bled ms serum 1/1,000 0.163 0.162

For the direct binding EIA, briefly, the diluted sera were reacted withmicrotiter wells directly coated with 100 ul of 3 ug/ml in PBS of p24M(i.e., p24 from group M) or rp240 (i.e., recombinant p24 from group)antigen or mixture of p24M and rp240 and then blocked with 2% bovineserum albumin (BSA) in PBS. After 1 hour incubation at room temperature(RT) on a mirotiter plate shaker (Lab-Line Instruments, Melrose, Ill.),the plate was washed 3 times with distilled water using a microtiterplate washer (Skanwash, Skatron Instruments, Sterling, Va.). One hundredul of 0.2 ug/ml of goat ant-mouse IgG+IgM-Horseradish Peroxidase (HRPO)(KPL, Gaithersburg, Md.) conjugate were added to each well of the plate.After incubating 30 minutes at RT, the plate was washed 3 times (asabove). Enzyme substrate o-phenylenediamine:2HCl (OPD) solution wasadded to each well to develop a color reaction for 5 minutes in the darkat RT. The reaction was stopped by addition of 1N H₂SO₄ into each well.The plate was read at A490 nm in a microtiter plate reader (Titertekmultiwell EIA reader, ICN, Huntsville, Ala.).

For the indirect sandwich EIA, a microtiter wells were coated with 100ul per well of 10 ug/ml in PBS of goat anti-mouse IgG+M antibodies (KPL)overnight at 2-8C. The plate was washed 3 times with distilled waterusing a plate washer (Skanwash, Skatron Instruments, Sterling, Va.) andthen blocked with 2% BSA in PBS for 30 minutes at RT. One hundred ulportions of culture fluids were added to the wells, and the plate wasincubated for 1 hour at RT on a plate shaker. Anti-p24 antibodiessecreted in culture fluids were captured by goat anti-mouse IgG+M coatedon solid phase. After washing, 100 ul portions of 100 pg/ml of HIV-1viral lysate were added into each well and the plate was incubated forone hour at RT on a plate shaker. After washing, 100 ul portions of 0.5ug/ml of rabbit anti-p24 antibodies were added into each well and theplate was incubated for one hour at RT on a plate shaker. After washing,100 ul of 0.2 ug/ml of goat anti-rabbit IgG-HRPO (KPL) were added toeach well, and the plate was incubated for 30 minutes at RT. After thefinal washing, chromogen OPD was added as described above.

Example 4 Cell Fusion

Three days after the pre-fusion antigen boost, mice were sacrificed andtheir spleens were disrupted to single cells. The single cellsuspensions were treated with 0.83% NH₄Cl to remove red blood cells, andthen mixed with SP2/0 cells at a 10:1 ratio of SP2/0:spleen cells. Themixed cells were centrifuged, washed once with serum-free medium, thencentrifuged again. The supernatant was removed from the cell pellet. Thefusogen, polyethylene glycol (PEG), was used to form hybrids of immunespleen cells with myeloma cell line SP2/0 (HPRT neg.) [Kohler andMilstein, Nature (1975) 256:494, and reviewed in Monoclonal HybridomaAntibodies: Techniques and Applications ed. Hurrell (CRC Press, Inc.,19820]. Briefly, fusion of spleen cells and SP2/0 cells was accomplishedby exposing the pellet to 40% PEG (M.W. 1450, American Type CultureCollection, Manassas, Va.) in serum-free Iscoe's Modified Dulbecco'sMedium (IMDM) for two minutes. The PEG and cell suspension was dilutedslowly by the addition of 20 ml of serum free IMDM over a period of fiveminutes, followed by collection of the cells by centrifugation. Thesupernatant was decanted and replaced with 30 ml IMDM containing 20%fetal bovine serum (Hyclone, Logan, Utah) with HAT (hypozanthine,aminopterin, and thymidine) to select for hybridomas. Spleen cells fromone nonimmunized BALB/c mouse also were added as a feeder layer. Thecells were plated at 0.1 ml/well in three 96 well tissue culture plates.Three days later an additional 0.1 ml of HAT media was added to eachwell. At weekly intervals thereafter, one half of the media was replacedwith IMDM containing 20% fetal bovine serum with HAT, and hybrids wereallowed to grow an additional 7-14 days.

Some of the hybrids were composed of spleen cells, making antibody toHIV-1, fused with SP2/0 cells. Briefly, the fusogen promotes fusion ofspleen cell and SP2/0 cell membranes, forming a heterokaryon containingnuclei of both cells. Eventually, the dissimilar nuclei fuse producing asingle nucleus capable of synchronous mitosis. As the fused cellsdivide, the hybrid stabilizes by losing chromosomes of each nucleus. Thefused cells were plated into multiple 96 well plates at 10⁵ to 10⁶ cellsper well. Hybrid cells formed from SP2/0:spleen cell fusions wereselectively propagated by culturing in HAT medium. All unfused SP2/0 orSP2/0:SP2/0 fused cells were prevented from growing by aminopterin, andunfused spleen cells or spleen:spleen fused cells died off in culture.Only spleen cell:SP2/0 hybrids will grow in the HAT selection medium.

Example 5 Screening, Cloning and Characterization of p24 MonoclonalAntibodies

After 10-14 days, culture fluids from wells containing hybridoma cellgrowth were screened for antibody to HIV-1 p24. The indirect EIA wasused to screen the culture fluids from cell fusions #103, #108, #115B,and #117. In order to select anti-HIV core protein monoclonal antibodieswith high affinity, a direct sandwich EIA assay was also utilized toscreen potentially useful clones from cell fusions #120A and cloning offusions #115B and 120A. Both direct and indirect EIAs are described inthe section of antisera titer assessment of Example 2. The primaryscreening data from the hybridomas described in this application areshown in Table 2a and 2b. TABLE 2a Primary Fusion screened by indirectbinding EIA A490 nm reading Hybrid # HIV Ag used Sample Blank control103-350 p24-M lysate 0.921 0.030 108-394 p24-M lysate 0.497 0.000115B-151 p24-M lysate 0.662 0.012 115B-303 p24-M lysate 0.467 0.003117-289 p24-M lysate 0.295 0.000

TABLE 2b Primary Fusion screened by direct sandwich EIA A490 nm readingNegative Hybrid # HIV Ag used Sample control 120A-270 p24-M lysate 0.501−0.011

Hybridomas showing strong positive signal in the primary screening EIAswere transferred into 24-well plates for cell expansion. Culture fluidswere again assayed for the presence of anti-p24 antibody. Anti-p24positive hybrids were further expanded in T25 flask for cloning bylimiting dilution. Each expanded hybrid was plated in a 96-well plate ata dilution of 105 to 106 and allowed to grow 10-21 days. Culture fluidsfrom limiting dilution were assayed for the presence of anti-p24antibody. The hybridoma designation is based on a numbering system using3 numbers: the first being the fusion number, the second is the parentalhybrid number and the third is the subclone number. Each 96-well tissueculture plate is sequentially numbered 1 to 96. For example, hybridoma#103-350-474 originates from the 103^(rd) fusion. The parental hybrid is#350 as it derives from the 3^(rd) fusion plate in well #50. Thesubclone is #474 since it is from the 4^(th) cloning plate, well #74.The clones were obtained by limiting dilution using the guidelinesoutlined by J. W. Goding in Monoclonal Antibodies: Principles andPractice (Academic Press, N.Y., 1983). The primary cloning data for thehybridomas described in this application are shown in Table 3a and 3b.TABLE 3a Primary clone screened by indirect binding EIA A490 nm readingClone # HIV Ag used Sample Blank control 103-350-474 p24-M lysate 0.4890.021 108-394-470 p24-M lysate 0.466 0.000

TABLE 3b Primary clone screened by direct sandwich EIA A490 nm readingClone # HIV Ag used Sample Blank control 115B-151-423 p24-M lysate 0.8460.000 115B-303-620 p24-M lysate 0.991 0.006 117-289-555 p24-M lysate0.830 0.011 120A-270-108 p24-M lysate 0.371 −0.021

The isotypes of anti-p24 Mabs were determined with the SBA ClonotypingSystem (Southern Biotechnology Associates, Inc., Birmingham, Ala.).Briefly, microtiter wells plate were coated with 100 ul portions of goatanti-mouse IgG+M antibodies (KPL) for 18-24 hours at 2-8C. The wellswere washed and blocked with 2% BSA in PBS for 30 minutes at RT. Afterwashing, 100 ul portions of culture fluids were added into the wells andincubated for 2 hour at RT on a plate shaker. After washing, 100 ulportions of rabbit anti-mouse isotype-specific antibodies were added tothe wells and incubated for one hour at RT on a plate shaker. Afterwashing, 100 ul of goat anti-rabbit IgG-HRPO (KPL) were added to thewells and incubated for another 30 minutes at RT on a plate shaker.After the final washing, chromagen OPD was added as described above. Theisotypes of Mabs 103-350-474, 108-394-470, 115B-151-423, 115B-303-620,117-289-555, and 120A-270-108 are summarized in Table 4. TABLE 4Monoclonal antibody isotypes Mab ID Isotype Light chain 103-350-474IgG2a kappa 108-394-470 IgG2b kappa 115B-151-423 IgG1 kappa 115B-303-620IgG2b kappa 117-289-555 IgG1 kappa 120A-270-108 IgG1 kappa

The specific reactivities of Mabs to HIV-½ antigens were tested in thedirect sandwich EIA as described in the previous section. The resultsare summarized in Table 5. TABLE 5 Reactivities of anti -HIV-1 p24 Mabswith HIV-1/2 p24/p26 Reactivity with Mab ID p24-M p24-O lysate HIV-2lyaste 103-350-474 ++ ++ ++ 108-394-470 ++ ++ + 115B-151-423 ++ ++ ++115B-303-620 ++ ++ ++ 117-289-555 ++ ++ ++ 120A-270-108 ++ ++ ++++ = Very Strong

In view of the results presented in Table 5, all of the monoclonalantibodies of the present invention react with HIV-½ p24/p26 antigens.(HIV-1 group O and HIV-2 NIH-Z viral lysates were purchased from ABI(Gaithersburg, Md.).)

Example 6 Antibody Production and Purification

In order to produce large amounts of Mabs for further characterizationand testing, anti-p24 hybridoma cell lines were further expanded in T250flasks and weaned to serum free media, H-SFM (Life Technologies, GrandIsland, N.Y.) When the hybridoma cell lines were adapted to H-SFM, theywere seeded in roller bottles for large scale antibody production.Culture fluids were harvested from the roller bottles and concentratedby a filtration system. The roller bottle derived antibody was purifiedon a Protein A column from PerSeptive Biosystems (Cambridge, Mass.).

Example 7 Affinity Measurement of Anti-p24 Monoclonal Antibodies

Affinities of purified anti-p24 Mabs 103-350-474, 108-294-470,115B-303-620, 115B-151-423, 117-289-555, and 120A-270-108 were measuredby a surface plasma resonance (SPR) based BIAcore immunosensorinstrument (Pharmacia, Uppsala, Sweden). Briefly, goat anti-mouse IgG(Fc) antibodies were covalently coupled to amino-sensor chips by EDACchemistry. Each mouse IgG monoclonal antibody was injected into thesensor chip and captured by the immobilzed goat anti-mouse IgGantibodies. The unbound mouse monoclonal antibody was washed away fromthe chip. A baseline measurement of surface plasma resonance (SPR)signal was recorded for each monoclonal. When purified HIV-1 p24 proteinwas injected into the sensor chip and reacted with anti-p24 monoclonalantibody, SPR signal started to increase. The slope of binding curve wasproportional to the association constant of each monoclonal antibody.After binding was achieved, a wash step was introduced. The dissociationrate of p24 from anti-p24 monoclonal antibody was proportional to thedecrease of SPR signal. After each cycle of measurement, HCl buffer wasapplied into the sensor chip to remove anti-p24 monoclonal antibody fromthe sensor chip for the next measurement. Based upon the on-rate andoff-rate SPR signals, association (Ka), dissociation (Kd), and relativeaffinity (K) constants of each monoclonal antibody were determined. Thedata are summarized in Table 6. TABLE 6 Binding constants Clone #Ka(M-1s-1) Kd(s-1) K(M-1) 103-350-474 8.3 × 10e5 5.1 × 10e−4 1.6 × 10e9108-394-470 8.1 × 10e5 4.2 × 10e−4 1.9 × 10e9 115B-151-423 1.3 × 10e62.0 × 10e−4 6.5 × 10e9 115B-303-620 8.3 × 10e5 2.6 × 10e−4 3.2 × 10e9117-289-555 3.5 × 10e5 3.3 × 10e−4 1.1 × 10e9 120A-270-108 8.1 × 10e58.8 × 10e−4 9.2 × 10e9

Example 8 Epitope Mapping of p24 Monoclonal Antibodies

Epitopes on HIV-1 p24 recognized by mouse monoclonal antibodies wereidentified using two sets of thirteen p24 synthetic peptides (FIG. 1 a).Peptide design was based on the three dimensional structure of p24antigen (Gitti, et al., Science 273: 231 (1996); Gamble, et al., Science278: 849 (1997)) in order to present selected, uninterrupted regions ofhelical structure (in vivo) that might be unique to shared epitopes.These peptides covered all helical regions (A-J) on the core proteins.Monoclonal antibodies were reacted against both group M (clade B) andgroup O (Ham112) peptides. Peptides were designated M1 to M13representing HIV-1 group M clade B p24, or O1 to O13 representing HIV-1group O (Ham 112 isolate) p24. Each peptide also contained an additionalcysteine on its C-terminus which was reacted with maleimide-modifiedKeyhole Lympet Hemocyanin (KLH) to form two series of KLH conjugatedpeptides in addition to unconjugated peptides. KLH conjugated peptideswere generated in order to help stabilize and present conformationalstructures that might be essential for epitope presentation andrecognition (monoclonal binding). KLH conjugated peptides weredesignated as KM1 to KM13 for group M peptides or KO1 to KO13 for groupO peptides.

Binding of monoclonal antibodies (Mabs) to sets of synthetic peptideswas determined by an indirect ELISA assay. Briefly, free or KLHconjugated synthetic peptides were coated on the wells of microtiterplates. The peptide coated wells were incubated with Mabs prepared to aconcentration of approximately 1 ug/ml. Bound Mabs were detected byenzyme or acridinium-labeled goat anti-mouse IgG antibodies.Representative data are depicted in FIGS. 2 a and b. Epitopes recognizedby Mab 103-350-474, 117-289-555, 115B-303-620 and 120A-270-108 wereidentified as follows:

Monoclonal antibody 103-350-474 specifically bound to the KLH conjugatedgroup M and group O peptides #4 (KM4/KO4), which corresponds to thehelix D region of p24. The epitope is linear with apparent secondaryconformational requirements. Little or no consistent binding wasdetected when 103-350-474 was reacted against free (un-conjugated)peptides. In contrast, when peptides were conjugated to a carrierprotein (KLH) in order to promote secondary conformational peptidestructures, 103-350-474 consistently exhibited strong (high S/N) bindingagainst KM4/KO4 peptides. The epitope appears to be linear because theantibody binds small synthetic peptides, but the optimal epitope mayrequire specific secondary helical structures. Therefore, the epitope ismost broadly defined as comprising amino acids 63-89, and most narrowlyestimated as requiring amino acids 63-80, which map to the helix-Dregion. Further, cross reactivity of the antibody between M and O p24indicates that the p24 regions of greatest sequence homology in helix-Dare most likely involved in forming the epitope. The residues in boldare most likely key to forming the epitope, but secondary structureinvolving or requiring neighboring amino acids cannot be excluded. (K)M4peptide 63-89: C QAAMQ MLKET INEEA AEWDR VHPVH AG (SEQ ID NO:1) (K)O4peptide 63-89: C QGALQ VLKEV INEEA ADWDR SHPPV VG (SEQ ID NO:2)

Monoclonal antibody 117-289-555 specifically bound to both group M andgroup O M10/O10 peptides, which correspond to the helix H region, whichis part of the major homology region (MHR). The epitope appears to belinear because the antibody readily binds free (un-conjugated) M10/O10peptides. Binding to both group M and group O p24 is expected. Theresidues in bold are likely key to forming the epitope. M10 peptide151-176: CLDIRQ GPKEP FRDYV DRFYK TLRAEQ (SEQ ID NO:3) O10 peptide152-177: CLDIKQ GPKEP PRDYV DRFYK TLRAEQ (SEQ ID NO:4)

Monoclonal antibody 115B-303-620 bound to M12 and O12 peptides, whichcorresponds to the helix J-K region of p24. The epitope appears to belinear based on the strong (high S/N) binding to free (un-conjugated)peptides of M12/O12. The residues in bold are likely key to forming theepitope, but secondary structure involving or requiring neighboringamino acids cannot be excluded. M12 peptide: CKTIL KALGP AATLE EMMTA(SEQ ID NO:5) O12 peptide: CKQIL KALGP GATLE EMMVA (SEQ ID NO:6)

Monoclonal antibody 120A-270-108 mapped to the helix H and MHR region ofp24 Similar to monoclonal antibody 117-289-555. However, 120A-270-108recognizes an epitope distinct from an epitope recognized by117-289-555. The significant difference between 117-289-555 and120A-270-108 is that 120A-270-108 only moderately bound to the KLHconjugated M10 peptide. No binding was detected when 120A-270-108 wasreacted against free (un-conjugated) peptides. Thus, the optimal epitopeof 120A-270-108 requires specific secondary or tertiary structures.Furthermore, 117-289-555 and 120A-270-108 belong to differentcompatibility groups because they bind simultaneously to core proteinswithout interference or competition from each other (see Table 7 below).TABLE 7 Sandwich formation of mAbs with p24proteins (both group M andgroup O) Signal labeled mAbs in solution phase mAbs in 103- 117- 115B-115B- 108- 120A- solid 350- 289- 303- 151- 394- 270- phase 474 555 620423 470 108 103-350- − + + + − − 474 117-289- + − + + + + 555115B-303- + + − + + + 620 115B- + + + − + + 151-423 108-394- − + + + − −470 120A-270- − + + + − − 108 120B-580- + + + − +/− − 106 helix AThe + sign indicates compatibility of paired mAbs binding to p24 antigensimultaneously

The compatibility study also showed that, unlike 117-289 which can pairwith either Mab 103-350 or 108-394 to form a sandwich, 120A-270 cannotform a sandwich with either Mab (Table 7). The compatibility dataclearly demonstrated that the epitope recognized by 120A-270 is distinctand different from the epitope recognized by 117-289. Mab 120A-270 mostlikely recognizes a conformational epitope, formed in part, by aminoacids 151-176.

Two Mabs 115B-151 and 108-394, fail to bind free synthetic peptides, and115B-151 weakly bound to one KLH coupled peptide (FIG. 2B). Failure tobind synthetic peptides indicated that these Mabs recognizeconformational epitopes on core antigen. Conformational epitopes areformed by contiguous amino acids brought together by the tertiary orquaternary folding of p24 antigen. A tertiary or quaternary structuredependent conformational epitope generally cannot be mimicked by smallsynthetic peptides.

A set of large overlapping p24 polypeptides were used to locateconformational epitopes recognized by the 115B-151 and 108-394.Overlapping p24 polypeptides were expressed in E. coli. (rproteins) fromplasmids carrying unique portions of p24 nucleotide sequence (deletionclones). Two sets of six deletion clones were designed based on thestructure of p24. Specific binding of monoclonal antibodies to p24polypeptides was determined using the Western blot method. Briefly, theexpressed (recombinant) p24 polypeptides in extracts of E. coli weresubjected to SDS-PAGE and transferred to nitrocellulose membranes. Themembranes containing electrophoreticaly separated proteins were reactedwith p24 Mabs (˜5 ug/ml concentration) and bound monoclonal antibodieswere detected by enzyme labeled goat anti-mouse IgG. FIG. 4 illustratesWestern blot results of Mabs 115B-151 and 108-394.

Monoclonal antibody 115B-151 specifically bound to group M polypeptidesF(1-172) and G(137-231) and group O polypeptides N(1-173) andO(138-232). The overlapping region (amino acids 137-172) between F and Gand N and O, may contribute to epitope recognized by 115B-151. This datais supported by weak but consistent binding of 115B-151 against the KLHconjugated synthetic peptides KM10 and KO10, which contain amino acids137-172 (FIG. 2B) Mab 115B-151 differed from 117-289 and 120A-270because (a) 115B-151 apparently required the epitope to be in a specificsecondary or tertiary conformation, (b) it was compatible with117-289/120A-270 to form a sandwich with p24 (Table 7), and (c)monoclonal antibodies against helix A were incompatible and stronglycompeted with 115B-151, but were compatible with 117-289. The strongcompetition of helix A directed monoclonal with 115B-151, in addition tothe weak binding against KLH conjugated peptides, may indicate that theoptimal epitope recognized by 115B-151 includes a portion of helix A inaddition to the minimal linear epitope identified within helix H.

Monoclonal 108-394 mapped to a conformational epitope formed within thefirst 172/173 amino acids of M/O p24. The epitope is non-linear becauseHIV-1 M and O synthetic peptides, free or conjugated to carrier proteinKLH, were unreactive with 108-394. In addition, 108-394 reacted onlywith the largest of the polypeptides (F 1-172/N 1-173) derived from M/Op24. Although M polypeptides C (1-65), E_(1-130), and I (60-150), andpolypeptides L (1-65), M (1-131), and Q (60-151) contain large segmentsof the same sequence found in polypeptides F (1-172) and N (1-173), anepitope recognized by 108-394 apparently was not formed from the shorterpolypeptides. These data are consistent with a conformational epitopeformed by a major portion of p24 comprising at least the first 172/173amino acids.

Based on the data from epitope mapping using synthetic peptides and p24deletion clones and the data from the p24 sandwich compatibility study,an epitope map of the six mAbs on the three dimensional structure of p24antigen is illustrated in FIG. 5. The structure of the p24 molecule isrepresented by two domains, the N-terminal domain (amino acids 1-151)and the C-terminal domain (amino acids 151-231). The structure of theintact p24 molecule has not been determined so the exact structuralrelationship between the two domains is not fully characterized.

The six mAbs mapped to p24 antigen in the following manner (FIG. 5):

Monoclonal antibody 103-350-474 binds a linear epitope located in thehelix D region of HIV-1 M and O p24. The epitope is most broadly definedas comprising amino acids 63-89, and is most narrowly estimated ascomprising amino acids 63-80 of group M and group O of the p24 antigen.

Monoclonal antibody 117-289-555 binds a linear epitope located in theMHR/helix H region of p24. The epitope is most broadly defined ascomprising amino acids 151-172 (M)/152-173 (O), and is most narrowlydefined as amino acids 162-172(M)/163-173(O) of p24 antigen.

Monoclonal antibody 115B-303-620 binds a linear epitope located in thehelical J-K regions of p24 antigen. The epitope is defined as aminoacids 198-217(M)/199-218(O) of the p24 antigen.

Monoclonal antibody 115B-151-423 binds a conformational epitope which ismost likely near the junction part of helix A and MHR/helix H regions ofp24.

Monoclonal antibody 108-394-470 binds a conformational epitope formedwithin the N-terminal domain (amino acids 1-151) of p24. Theconformational epitope is estimated to be near the junction part ofhelix D and helix A.

Monoclonal antibody 120A-270-108 binds a conformational epitope which isestimated to be near the junction part of helix D, helix A and MHR/helixH regions of p24.

Example 9 Preparation of Monoclonal Antibody Coated Microparticles

Carboxyl-modified latex (CML) microparticles at 1% solid (obtained fromBangs Laboratories, Fishers, Ind.) were activated by carbodiimide EDC[1-ethyl-3-3-dimethylaminopropyl) carbodiimide hydrochloride from SigmaChemicals, St. Louis, Mo.] at a molar ratio of EDC: carboxyl groups=10:1in 50 mM MES [2-(N-morpholino)ethane sulfonic acid] buffer pH 6.1 for 5minutes at RT on a end-over-end rotator (Roto-Torque, Cole ParmerInstruments, Vernon Hills, Ill.) set at high on scale S. Anti-core Mabwas added to EDC pre-activated CML microparticles at a ratio of 200 ugantibody/per ml of 1% solid microparticles for 4 hours at roomtemperature on an end-over-end rotator. Free reactants were washed awayusing Abbott diafiltration system (Abbott Laboratories, Abbott Park,Ill.) with a crossflow syringe membrane (0.2 um pore size and 12 cm²surface area, obtained from Spectrum, Laguna Hills, Calif.). Mab-coatedmicroparticles were overcoated with buffer containing 10 mM PBS and 5%BSA, 0.03% sodium azide for 1 hour at RT on a rotator. Mab-coated CMLmicroparticles were heat-stressed in a 45 C oven incubator for 20 hoursand then tested by an Abbott Prism Stand-alone instrument (AbbottLaboratories, Abbott Park, Ill.).

Example 10 Preparation of Acridinium-Labeled Antibody Conjugates

Anti-HIV core Mabs in PBS, pH 7.4 reacted withacridinium-N-hydroxysuciniimide (ACR—NHS) active ester at the molarratio of Mab:ACR-NHS=1:15 for 10 minutes at RT on an end-over-endrotator (Cole Parmer Instruments, Vernon Hills, Ill.). ACR-labeled-Mabconjugate was separated from free reactants by a G-25 Sephardex column(15 cm×1.5 cm) which was pre-equilibrated in PBS pH 6.3 containing 1%CHAPS. The elution peak of ACR-labeled-Mab was monitored by followingabsorbance at 280 nm using a spectrophotometer (Shimadzu UV-2101PC). Theconcentration of ACR-labeled-mAb protein=(OD280 nm-0.247×OD370 nm)/1.38.Molar ratios of ACR/mAb=[OD370 nm/(OD280 nm-0.247×OD370 nm)]×15.

Example 11 Prism Immunoassay Methods and Preparation

Anti-p24 mAb-microparticles (concentrate stock) were diluted inuParticle diluent (10 mM PBS, pH 6.5 containing 5% calf serum, 7.5%sucrose, 50 mM EDTA, 0.1% Tween 20, and 0.1% proclin). Anti-p24 mAb-ACRconjugates were diluted in conjugate diluent (10 mM PBS, pH 6.3containing 40 mM EDTA, 5% calf serum, 0.5% Triton, and 0.1% proclin).Two wash buffers were used in the assay. Transfer wash buffer contained25 mM MES pH 5.7, 150 mM NaCl, 4% Triton X-100, 1% Tween 20, 0.001% PEG,0.1% proclin, and 0.001% antifoam. Conjugate wash buffer contained 10 mMCAPS, pH 9.9, 150 mm NaCl, 5% Triton X-100, 0.1% proclin, and 0.001%antifoam. HIV-1 p24-M, rp24-O, HIV-2 rp26, and HIV-2 viral lysate werediluted in HIV-½ negative human plasma. Normal human plasma was alsoused as a negative control.

Assay Procedure:

Briefly, all reagents were warmed up to room temperature before primingthe instrument. Transfer wash buffer, conjugate wash buffer, anti-p24mAb-ACR conjugate, and activator were connected to a proper reagentline. The reagents were primed two times and any air bubbles trapped inthe reagent lines were tapped away. One hundred ul of samples and 50 ulof specimen diluent (25 mM PBS pH 6.5, 1% Triton X-100, 0.4% Tween 20,20 mM EDTA, and 0.1% proclin) were added into channel A and B reactionwells of sample tray manually. The trays were loaded on the instrumentand moved constantly through the channel during the assay steps. Whenthe sample tray moved to the microparticle station, 50 ul of anti-p24mAb-coated microparticles were added into each reaction well. The restof the assay steps were performed automatically by the instrument. Thechannel temperature of the instrument was maintained at 37 C. after 18minutes incubation at 37 C, the sample tray was moved to the transferwash station. The mixture of sample and uPrticles were flushed onto aglass fiber matrix by transfer buffer and washed 2 times with transferbuffer. Fifty ul of anti-p24 mAb-ACR conjugate were added onto the glassfiber matrix. After 20 minutes incubation, the sample tray was moved tothe conjugate wash station. Unbound conjugates were washed away byconjugate wash buffer. After the conjugate wash, the sample tray wasmoved to the activator station. Fifty ul of activator (mixture ofhydrogen peroxide and sodium hydroxide) were applied to the matrix. Thechemiluminescence light signal was read by a photomultiplier tubedetector.

Example 12 Equivalent Detection of HIV Core Proteins (Antigens) UsingTwo Monoclonal Antibodies

Equivalent, quantitative core antigen sensitivity using compatible pairsof high affinity monoclonals was demonstrated in a two-step,chemiluminescent, “sandwich” immunoassay run on an Abbott Prismstandalone instrument (Abbott Laboratories, Abbott Park, Ill.). Bycombining 120A-270-108 coated microparticles (0.066% solid) with115B-151-423-ACR conjugate at 60 ng/ml, equivalent detection of HIV-1group M, HIV-1 group O, and HIV-2 core proteins was achieved. The lowestlimits of detection for HIV-1 group M p24 was estimated at 0.3 pg/ml(FIG. 6), 0.3 pg/ml for HIV-1 group 0 rp24 (FIG. 7), and 1.0 pg/ml forHIV-2 rp26 (FIG. 8). Only a small (3.3 folds) differences inquantitative sensitivity was detected between HIV-1 and HIV-2.Equivalent sensitivity across three related but non-indentical coreantigens strongly argues that the unusually high Keq of these monoclonalantibodies are directed toward shared epitopes, not cross-reactiveepitopes. Affinity of these Mabs against core antigens (HIV-1 M, O, andHIV-2) must be nearly equal because of the near equivalent bindingkinetics against all three core antigens. Generally, Keq decreases whenMabs are reacted against cross-reactive epitopes, indicated by markedlylower quantitative sensitivity for the cross-reactive antigen comparedto the native (immunogen) antigen. Further, the small differencesbetween quantitation of HIV-1 and HIV-2 core proteins related herein mayrelate more to the methods and (error around the methods) used toquantitate the proteins for the studies.

1. A monoclonal antibody which binds to a shared epitope of HumanImmunodeficiency Virus-1 protein p24 and Human Immunodeficiency Virus-2protein p26, wherein said monoclonal antibody is 115B-151.
 2. (canceled)3. A hybridoma cell line which secretes a monoclonal antibody whichbinds to a shared epitope of Human Immunodeficiency Virus-1 protein p24and Human Immunodeficiency Virus-2 protein p26, wherein said cell lineis A.T.C.C. Deposit No. PTA-2809. 4-16. (canceled)
 17. A kit fordetermining the presence of one or more antigens selected from the groupconsisting of HIV-1 antigen and HIV-2 antigen in a test samplecomprising: (a) at least one monoclonal antibody which binds to a sharedepitope of Human Immunodeficiency Virus-1 protein p24 and HumanImmunodeficiency Virus-2 protein p26 wherein said at least onemonoclonal antibody is 115B-151; and (b) a conjugate comprising anantibody attached to a signal generating compound capable of generatinga detectable signal.
 18. (canceled)
 19. The kit of claim 17 wherein saidantibody of (b) is selected from the group consisting of 120A-270,117-289, 103-350, 115B-303 and 108-394.
 20. A diagnostic reagentcomprising at least one monoclonal antibody wherein said at least onemonoclonal antibody is 115B-151. 21-28. (canceled)